Obesity results from dysregulation of central and peripheral systems governing appetite and energy expenditure. These systems are made up of complex interactions between central centers in the hypothalamus and brainstem, adipocyte-derived proteins, and gut-derived proteins. Ghrelin is a recently described gut hormone originally discovered as an endogenous ligand for the growth hormone secretagogue receptor. In animals, ghrelin is a potent orexigen that can induce obesity and in humans has also been shown to stimulate appetite. Ghrelin is unique among known hormones in that it has an acyl group attached to a protein backbone, and this acyl group must be present for normal activity. Ghrelin levels are acutely suppressed by meals and basal levels are linked with insulin resistance. Several studies have shown differences in ghrelin suppressibility by meals that vary in macronutrient content. These differences could occur in response to a number of co-secreted hormones (including insulin, glucagon-like peptide 1, or peptide YY), and have important implications for the role of ghrelin in maintenance of the obese state. Previous studies have examined ghrelin regulation by predominantly measuring total immunoreactive levels, which includes both active and inactive forms of ghrelin. However, changes in total ghrelin may not reflect effects on active ghrelin levels;and few studies have attempted to characterize the structure of ghrelin protein (length of the protein backbone, types of fatty acid ligands), which could also affect ghrelin receptor signaling. Unique in subjects with known genetic obesity, those with Prader-Willi Syndrome (PWS) have found to have elevated levels of total ghrelin. Depending on the structure of this ghrelin, these high levels may be contributing to the voracious appetite characteristic of this syndrome. Studies proposed here, therefore, include the characterization of ghrelin's structure in PWS and controls to provide insight into its biological activity and cellular regulation, clarification of the nutrient regulation of ghrelin levels and structure in these groups, determine the effects of specific postprandial hormones on ghrelin levels and structure, and whether improvement in insulin sensitivity plays an independent role in the determining basal and meal-related ghrelin suppression in subjects with impaired glucose tolerance. In summary, the overall goals of this proposal are to study the effect of nutrient and pharmacological regulation on both ghrelin levels and structure using state-of-the-art methods. Results from these studies will broaden our understanding of the brain-gut axis involved in body weight regulation by clarifying the regulation of ghrelin levels and structure in lean and obese individuals.